Erve J C, Amarnath V, Graham D G, Sills R C, Morgan A L, Valentine W M
Department of Pathology and Center in Molecular Toxicology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2561, USA.
Chem Res Toxicol. 1998 May;11(5):544-9. doi: 10.1021/tx980007i.
CS2, a known neurotoxicant, is used in the viscose production of rayon and is also a decomposition product of N, N-diethyldithiocarbamate, a metabolic product of the drug disulfiram used in alcohol aversion therapy. Previous in vitro investigations have demonstrated the ability of CS2 to cross-link proteins through thiourea, dithiocarbamate ester, and disulfide structures. Although in vivo studies have supported protein cross-linking as both a mechanism of neurotoxicity and a potential biomarker of effect, the chemical structures responsible for CS2-mediated protein cross-linking in vivo have not been elucidated. In the present study, the structure of one type of stable protein cross-link produced on erythrocyte spectrin by CS2 in vivo is determined. Rats were exposed to 50, 500, and 800 ppm CS2 for 13 weeks by inhalation or to 3 mmol/kg N,N-diethyldithiocarbamate administered orally on alternating days for 8 weeks. Erythrocyte spectrin preparations from control and exposed rats were hydrolyzed using 6 N HCl and separated by size-exclusion chromatography. The fraction that coeluted with the synthetic deuterated lysine-lysine thiourea internal standard was derivatized with 3-[4'-[(N,N,N-trimethylamino)ethylene]phenyl] 2-isothiocyanate and analyzed by liquid chromatography tandem mass spectrometry using selected reaction monitoring detection. Lysine-lysine thiourea was detected in spectrin preparations obtained from CS2-treated rats at 500 and 800 ppm and N, N-diethyldithiocarbamate-treated rats, but not from controls. These results establish that CS2-mediated protein cross-linking occurs in vivo through the generation of Lys-Lys thiourea and that diethyldithiocarbamate can, through in vivo release of CS2, produce the same cross-linking structure. This observation supports the utility of cross-linking of peripheral proteins as a specific dosimeter of internal exposure for CS2 and provides a mechanistic explanation to account for the high-molecular-weight neurofilament protein species isolated from rats exposed to CS2 or N, N-diethyldithiocarbamate.
二硫化碳（CS2）是一种已知的神经毒物，用于人造丝的粘胶生产，也是戒酒硫（一种用于戒酒疗法的药物）的代谢产物N，N - 二乙基二硫代氨基甲酸盐的分解产物。先前的体外研究表明，CS2能够通过硫脲、二硫代氨基甲酸盐酯和二硫键结构使蛋白质交联。尽管体内研究支持蛋白质交联是神经毒性机制和潜在效应生物标志物，但CS2在体内介导蛋白质交联的化学结构尚未阐明。在本研究中，确定了CS2在体内使红细胞血影蛋白产生的一种稳定蛋白质交联的结构。大鼠通过吸入暴露于50、500和800 ppm的CS2中13周，或每隔一天口服3 mmol/kg的N，N - 二乙基二硫代氨基甲酸盐，持续8周。使用6 N盐酸水解来自对照和暴露大鼠的红细胞血影蛋白制剂，并通过尺寸排阻色谱法进行分离。与合成的氘代赖氨酸 - 赖氨酸硫脲内标共洗脱的馏分用3 - [4'-[(N，N，N - 三甲基氨基)乙烯]苯基] 2 - 异硫氰酸酯衍生化，并使用选择反应监测检测通过液相色谱串联质谱法进行分析。在从500和800 ppm CS2处理的大鼠以及N，N - 二乙基二硫代氨基甲酸盐处理的大鼠获得的血影蛋白制剂中检测到赖氨酸 - 赖氨酸硫脲，但在对照中未检测到。这些结果表明，CS2介导的蛋白质交联在体内通过赖氨酸 - 赖氨酸硫脲的生成而发生，并且二乙基二硫代氨基甲酸盐可通过体内释放CS2产生相同的交联结构。这一观察结果支持外周蛋白交联作为CS2体内暴露特异性剂量计的实用性，并为解释从暴露于CS2或N，N - 二乙基二硫代氨基甲酸盐的大鼠中分离出的高分子量神经丝蛋白种类提供了机制解释。
